PROJECT SUMMARY Environmental fluctuations in the human oral cavity favor an increase in the number of cariogenic bacteria, including Streptococcus mutans, one of the etiological agent of human dental caries. Dental caries is the most common chronic disease in children, can greatly impact quality of life, and represents 5-10% of healthcare expenditures in industrialized nations. One attractive strategy towards the growth inhibition of cariogenic bacteria is an oral bacteria probiotic approach that seeds oral biofilms with beneficial commensals that can moderate plaque acidification and interfere with the colonization and virulence of caries pathogens such as S. mutans. Thus, a better understanding of the mechanisms by which health-associated bacteria antagonize and compete with cariogenic bacteria is needed. The virulence of S. mutans resides in three core attributes: its ability to form biofilms on the tooth surface, to produce large quantities of organic acids from a wide range of carbohydrates, and its ability to tolerate environmental stressors. Of interest, a very strong correlation exists between stress tolerance, biofilm formation and natural genetic competence [or the ability to uptake external DNA from the environment]. Recently, our group developed a Tn-seq library in S. mutans for discovering regions of the genome that are required for the survival of bacteria in different environments. Interestingly, when the library was input into an in vivo rodent model of oral infection, genes involved in genetic competence (comR, comX) showed an enhancement in recovery suggesting that their deletion led to an increase in fitness. This contrasts with the fact that many of the competence genes are highly conserved across human isolates of S. mutans signifying an evolutionary pressure to keep the competence pathway intact. One hypothesis is that the mouse caries model may not fully recapitulate the selective pressures found in the human host; the competence genes may play an important role in competition with commensal bacteria that is either suppressed or not present in the mouse model used. Therefore, we purpose two critical aims of this grant application to evaluate the impact of competence development in the ecology and development of oral biofilms. In Aim 1, we will test a library of novel streptococcal species that were isolated from dental plaque of subjects of known caries status that display a wide range of antagonistic capabilities towards S. mutans and determine if the S. mutans cell-cell signaling response is altered when these strains are cultured together. In Aim 2, we will elucidate how modulation of S. mutans cell-cell signaling either fosters or inhibits the establishment and outgrowth of S. mutans by monitoring the fitness of mixed-species biofilm populations, changes in biofilm architecture, and alterations in the spatial arrangement between competing strains at selected time points. Together, the results found here will establish a new foundation for the design and discovery of new and novel anti-caries therapies, and allow us to determine the ecological consequences of selective inhibition in oral biofilm populations.